The CDK4/6 Inhibitor Palbociclib Synergizes with ATRA to Induce Differentiation in AML.

Differentiation therapy based on ATRA almost cured acute promyelocytic leukemia (APL). However, it is disappointing that ATRA is not effective against other acute myeloid leukemia (AML) subtypes. Developing new and effective anti-AML therapies that promote leukemia differentiation is necessary. The CDK4/6-cyclin D pathway is a key initiator of the G1-S phase transition, which determines cell fate. Herein, we investigated whether the CDK4/6 inhibitor palbociclib would synergize with ATRA to promote leukemia differentiation in vitro and in vivo. Our findings revealed that CDK4/6-cyclin D pathway genes were aberrantly expressed in AML, and we observed that palbociclib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes indicative of myeloid differentiation. The combination of palbociclib and ATRA attenuated AML cell expansion in vivo. These enhanced differentiation effects may be associated with the regulation of transcription factors, including RARα, E2F1, and STAT1. Overall, our findings demonstrate that CDK4/6 inhibition sensitizes AML cells to ATRA and could guide the development of novel therapeutic strategies for patients with AML.

Genome-wide identification of the melon (Cucumis melo L.) response regulator gene family and functional analysis of CmRR6 and CmPRR3 in response to cold stress.

The response regulator (RR) gene family play crucial roles in cytokinin signal transduction, plant development, and resistance to abiotic stress. However, there are no reports on the identification and functional characterization of RR genes in melon. In this study, a total of 18 CmRRs were identified and classified into type A, type B, and clock PRRs, based on phylogenetic analysis. Most of the CmRRs displayed tissue-specific expression patterns, and some were induced by cold stress according to two RNA-seq datasets. The expression patterns of CmRR2/6/11/15 and CmPRR2/3 under cold treatment were confirmed by qRT-PCR. Subcellular localization assays indicated that CmRR6 and CmPRR3 were primarily localized in the nucleus and chloroplast. Furthermore, when either CmRR6 or CmPRR3 were silenced using tobacco ringspot virus (TRSV), the cold tolerance of the virus-induced gene silencing (VIGS) melon plants were significantly enhanced, as evidenced by measurements of chlorophyll fluorescence, ion leakage, reactive oxygen, proline, and malondialdehyde levels. Additionally, the expression levels of CmCBF1, CmCBF2, and CmCBF3 were significantly increased in CmRR6-silenced and CmPRR3-silenced plants under cold treatment. Our findings suggest that CmRRs contribute to cold stress responses and provide new insights for further pursuing the molecular mechanisms underlying CmRRs-mediated cold tolerance in melon.

An Intrabody against B-Cell Receptor-Associated Protein 31 (BAP31) Suppresses the Glycosylation of the Epithelial Cell-Adhesion Molecule (EpCAM) via Affecting the Formation of the Sec61-Translocon-Associated Protein (TRAP) Complex.

The epithelial cell-adhesion molecule (EpCAM) is hyperglycosylated in carcinoma tissue and the oncogenic function of EpCAM primarily depends on the degree of glycosylation. Inhibiting EpCAM glycosylation is expected to have an inhibitory effect on cancer. We analyzed the relationship of BAP31 with 84 kinds of tumor-associated antigens and found that BAP31 is positively correlated with the protein level of EpCAM. Triple mutations of EpCAM N76/111/198A, which are no longer modified by glycosylation, were constructed to determine whether BAP31 has an effect on the glycosylation of EpCAM. Plasmids containing different C-termini of BAP31 were constructed to identify the regions of BAP31 that affects EpCAM glycosylation. Antibodies against BAP31 (165-205) were screened from a human phage single-domain antibody library and the effect of the antibody (VH-F12) on EpCAM glycosylation and anticancer was investigated. BAP31 increases protein levels of EpCAM by promoting its glycosylation. The amino acid region from 165 to 205 in BAP31 plays an important role in regulating the glycosylation of EpCAM. The antibody VH-F12 significantly inhibited glycosylation of EpCAM which, subsequently, reduced the adhesion of gastric cancer cells, inducing cytotoxic autophagy, inhibiting the AKT-PI3K-mTOR signaling pathway, and, finally, resulting in proliferation inhibition both in vitro and in vivo. Finally, we clarified that BAP31 plays a key role in promoting N-glycosylation of EpCAM by affecting the Sec61 translocation channels. Altogether, these data implied that BAP31 regulates the N-glycosylation of EpCAM and may represent a potential therapeutic target for cancer therapy.

Knockdown of BAP31 Downregulates Galectin-3 to Inhibit the Wnt/ß-Catenin Signaling Pathway to Modulate 5-FU Chemosensitivity and Cancer Stemness in Colorectal Cancer.

Increased stemness is causally linked to the development of chemoresistance in cancers. B-cell receptor-associated protein 31 (BAP31) has been identified to play an oncogenic role in many types of cancer. However, the role of BAP31 in 5-fluorouracil (5-FU) chemosensitivity and stemness of colorectal cancer (CRC) is still unknown. The aim of this study was to investigate the biological function and molecular mechanism of BAP31 in regulating 5-FU chemosensitivity and stemness. The correlation between BAP31 expression and 5-FU chemosensitivity was examined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and colony formation assays. Cancer stemness was analyzed using tumor sphere formation and Western blot assays. Western blot and immunofluorescence analyses of the knockdown cell lines were performed to explore the possible mechanisms. Finally, we investigated the function of BAP31 by constructing xenograft nude mouse models in vivo. In this study, we demonstrated that BAP31 was increased in CRC cells, and knockdown of BAP31 reduced the half maximal inhibitory concentration (IC50) of 5-FU, while this effect was reversed by overexpression of BAP31. In addition, knockdown of BAP31 substantially reduced the stemness of CRC cells in vitro. Consistently, knockdown of BAP31 significantly suppressed the tumorigenicity and stemness of CRC in vivo. The functional study further suggested that knockdown of BAP31 downregulated galectin-3 to inhibit the accumulation of ß-catenin, which in turn repressed the transcription of downstream target genes (c-MYC, SOX2) of the Wnt/ß-catenin signaling pathway. Knockdown of BAP31 reduced stemness by inhibiting the Wnt/ß-catenin signaling pathway to increase 5-FU chemosensitivity. Importantly, intrabodies against BAP31 suppressed tumor growth and enhanced the antitumor effects of 5-FU in vivo. Therefore, using intrabodies against BAP31 may be a strategy for improving the antitumor effect of 5-FU in CRC.

Clinical implications of aberrant PD-1 expression for acute leukemia prognosis.

BACKGROUND: Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are the most common types of leukemia in adults with an overall poor prognosis. PD-1 alone or combined with other immune checkpoint blockade is a promising research direction for the treatment of acute leukemia (AL) patients. However, clinical Implications of aberrant PD-1 expression in peripheral CD4+ and CD8+ T lymphocytes of AML and ALL patients in assessing the prognosis of diseases, remains inconclusive. METHODS: In the present study, we used flow cytometry to evaluate PD-1 expression on the surface of CD4+ and CD8+ T lymphocytes in the peripheral circulation of AML and ALL patients and its clinical significance. A total of 53 AML patients, 44 ALL patients and 28 healthy controls were enrolled in this study and peripheral blood specimens were detected by flow cytometry. RESULTS: Our results indicated that percentages of CD4+ PD1+ and CD8+ PD1+ T lymphocytes in newly diagnosed and non-remission groups were significantly higher than healthy control both in AML and ALL patients. The high level of CD4+ PD1+ and CD8+ PD1+ T lymphocytes were respectively poor prognostic indicators of AML patients and ALL patients but had no significant correlation with most common clinical risks. CONCLUSIONS: Our findings show that aberrant PD-1 expression correlates with the prognosis of AL patient and may thus serve as poor prognostic indicators. Immunotherapy using PD-1 inhibitors may be a promising strategy for AML and ALL patients with peripheral circulating CD4+ PD1+ and CD8+ PD1+ T lymphocytes positively expressed, respectively.

Caged Luciferase Inhibitor-Based Bioluminescence Switching Strategy Enables Efficient Detection of Serum APN Activity and the Identification of Its Roles in Metastasis of Non-Small Cell Lung Cancer.

Bioluminogenic probes emerged as powerful tools for imaging and analysis of various bioanalyses, but traditional approaches would be limited to the low sensitivity during determine the low activity of protease in clinical specimens. Herein, we proposed a caged luciferase inhibitor-based bioluminescence-switching strategy (CLIBS) by using a cleavable luciferase inhibitor to modulate the activity of luciferase reporter to amplify the detective signals, which led to the enhancement of detection sensitivity, and enabled the determination of circulating Aminopeptidase N (APN) activity in thousands of times diluted serum. By applying the CLIBS to serum samples in non-small cell lung cancer (NSCLC) patients from two clinical cohorts, we revealed that, for the first time, higher circulating APN activities but not its concentration, were associated with more NSCLC metastasis or higher metastasis stages by subsequent clinical analysis, and can serve as an independent factor for forecasting NSCLC patients' risk of metastasis.

The BAP31/miR-181a-5p/RECK axis promotes angiogenesis in colorectal cancer via fibroblast activation.

Background: B-cell receptor-associated protein 31 (BAP31) has been recognized as a tumor-associated protein and has largely been shown to promote metastasis in a variety of cancers. Cancer metastasis arises through multistep pathways, and the induction of angiogenesis is shown to be a rate-limiting step in the process of tumor metastasis. Methods and results: This study explored the effect of BAP31 on colorectal cancer (CRC) angiogenesis by regulating the tumor microenvironment. First, exosomes from BAP31-regulated CRCs affected the transition of normal fibroblasts to proangiogenic cancer-associated fibroblasts (CAFs) in vivo and in vitro. Next, microRNA sequencing was performed to analyze the microRNA expression profile of exosomes secreted from BAP31- overexpressing CRCs. The results indicated that the expression of BAP31 in CRCs significantly altered the levels of exosomal microRNAs, such as miR-181a- 5p. Meanwhile, an in vitro tube formation assay showed that fibroblasts with high levels of miR-181a-5p significantly promoted endothelial cell angiogenesis. Critically, we first identified that miR-181a-5p directly targeted the 3'-untranslated region (3'UTR) of reversion-inducing cysteine-rich protein with kazal motifs (RECK) using the dual-luciferase activity assay, which drove fibroblast transformation into proangiogenic CAFs by upregulating matrix metalloproteinase-9 (MMP-9) and phosphorylation of mothers against decapentaplegic homolog 2/Mothers against decapentaplegic homolog 3 (Smad2/3). Conclusion: Exosomes from BAP31-overexpressing/BAP31-knockdown CRCs are found to manipulate the transition of fibroblasts into proangiogenic CAFs by the miR-181a-5p/RECK axis.

Palbociclib enhances the effect of doxorubicin-induced apoptosis in activated B-cell-like diffuse large B-cell lymphoma cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma around the world. While R-CHOP has significantly improved patient outcomes, a subset of patients still has poor outcome. Here, the oncogenic roles of cyclin dependent kinase 4/6 (CDK4/6)-Cyclin D (CCND) signaling axis in DLBCL and its potential mechanism were investigated to explore the possibility of targeting CDK4/6-CCND signaling axis for DLBCL therapy. The transcription levels, functional enrichment analysis, mutation analysis, and prognostic values were performed via the Oncomine, GEPIA, UALCAN, cBioPortal, and Metascape and GenomicScape databases. Expression of CDK4/6-CCND signaling axis in DLBCL patients and DLBCL cell lines was evaluated by qRT-PCR. Additionally, the impact of CDK4/6-CCND signaling axis on cell viability and apoptosis in DLBCL cell lines were evaluated in vitro . The transcription levels of CDK4/6-CCND signaling were increased in DLBCL patients. Meanwhile, in Gene Expression Omnibus dataset, the expression of CDK4 and CCND2 was higher in ABC-DLBCL, whereas the expression of CCND1 and CCND3 was higher in GCB-DLBCL. Moreover, according to the results of qRT-PCR, the expression of CDK4/6-CCND signaling axis in ABC-DLBCL cell line is higher than that in GCB-DLBCL cell lines. Prognostic analysis indicated that upregulation of CDK4, CCND2, and CCND3 was significantly associated with poor survival. Cell function experiments showed that palbociclib could enhance the apoptosis-promoting and cell viability-inhibiting effects of doxorubicin on ABC-DLBCL (SU-DHL-2) cells. Doxorubicin accumulation experiment showed that palbociclib promoted doxorubicin accumulation in ABC-DLBCL cells. Additionally, Western blot analysis demonstrated that palbociclib prevented antiapoptotic protein BCL2 expression in ABC-DLBCL cell line. Our study provides novel insights into targeted therapies for ABC-DLBCL patients.

Gut-derived fungemia due to Kodamaea ohmeri combined with invasive pulmonary aspergillosis: a case report.

BACKGROUND: Kodamaea ohmeri is a rare pathogen with high mortality and is found among blood samples in a considerable proportion; however, gastrointestinal infection of K. ohmeri is extremely rare. Invasive pulmonary aspergillosis is also an uncommon fungal; these two fungal infections reported concomitantly are unprecedented. CASE PRESENTATION: We described a case of a 37-year-old male who got infected with K. ohmeri and invasive pulmonary aspergillosis. We used the mass spectrometry and histopathology to identify these two fungal infections separately. For the treatment of K. ohmeri, we chose caspofungin. As for invasive pulmonary aspergillosis, we used voriconazole, amphotericin B, and then surgery. The patient was treated successfully through the collaboration of multiple disciplines. CONCLUSIONS: We speculate that the destruction of the intestinal mucosa barrier can make the intestine one of the ways for certain fungi to infect the human body.

Elevated circulating myeloid-derived suppressor cells associated with poor prognosis in B-cell non-Hodgkin's lymphoma patients.

INTRODUCTION: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population with the ability to suppress immune responses. MDSCs usually cluster in cancer, inflammation, and autoimmune diseases. Although there have been some studies on MDSCs in non-Hodgkin lymphoma (NHL), the correlation between the peripheral levels of MDSCs in patients with various subtypes of B cell NHL and clinical features and prognosis remains inconclusive. This study aimed at the issue. METHODS: 101 patients with B cell NHL and 15 age-matched healthy controls were included in this study. Flow cytometric detection of monocytic-MDSCs (M-MDSCs) and granulocytic-MDSCs (G-MDSCs) was done. RESULTS: In this study, we found that counts of circulating M-MDSCs and G-MDSCs were significantly increased in different clinical statuses of B-NHL patients compared to healthy controls. Similarly, a significant increase in the levels of M-MDSCs and G-MDSCs was found among the diverse types of B-NHL compared with healthy donors. Stratification studies indicated MDSCs expansion was closely associated with disease progression (tumor stage, LDH levels and B syndromes). Moreover, the overall survival time of patients with G-MDSCs (%) ≥ 98.70% was shorter than patients with G-MDSCs (%) < 98.70% in newly diagnosed B-NHL subgroup, meanwhile, there was a significant difference in survival of patients with M-MDSCs (%) ≥ 7.19% compared to patients with M-MDSCs (%) < 7.19% in relapsed B-NHL subgroup. CONCLUSION: Our results suggested that M-MDSCs and G-MDSCs may be a potential and efficient index to evaluate the prognosis of B-NHL patients.

A rapid and durable response to cabozantinib in an osimertinib-resistant lung cancer patient with MET D1228N mutation: a case report.

Osimertinib has efficacy superior to that of standard epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) for the first-line treatment of patients with EGFR-mutant advanced non-small cell lung cancer (NSCLC). However, patients treated with osimertinib eventually acquire drug resistance. MET missense mutations have been demonstrated to mediate resistance to MET-TKIs, such as crizotinib. But the role of MET missense mutations in mediating EGFR TKI resistance is undefined. With the increasing use of next-generation sequencing (NGS) at diagnosis, many mechanisms of acquired resistance have been discovered in patients with activated tyrosine kinase receptors. Herein, we report the first case of MET D1228N mutation mediating acquired resistance to osimertinib in a MET TKI-naïve NSCLC. The patient with advanced lung adenocarcinoma harboring EGFR exon 19 deletion initially responded to osimertinib with progression-free survival (PFS) lasting 11 months and then developed resistance with an acquired mutation of MET D1228N. Subsequently, combination therapy of cabozantinib and osimertinib was administrated to the patient, and her clinical symptoms were rapidly relieved within one week with good tolerance. She remained on the combined treatment for 10 months. Finally, she achieved an overall survival (OS) of 25 months. Based on our findings, patient with MET D1228N mutant lung adenocarcinoma clinically benefited from combinatorial therapy of cabozantinib and osimertinib after osimertinib resistance.

Detecting ALK Rearrangement with RT-PCR: A Reliable Approach Compared with Next-Generation Sequencing in Patients with NSCLC.

BACKGROUND: Precise detection of anaplastic lymphoma kinase (ALK) rearrangement guides the application of ALK-targeted tyrosine kinase inhibitors (ALK-TKIs) in patients with non-small-cell lung cancer (NSCLC). Next-generation sequencing (NGS) has been widely used in clinics, but DNA-based NGS used to detect fusion genes has delivered false-negative results. However, fusion genes can be successfully detected at the transcription level and with higher sensitivity using RNA-based reverse transcription polymerase chain reaction (RT-PCR). OBJECTIVE: This study compared the performance of RT-PCR and NGS in the detection of echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion in Chinese patients with NSCLC. METHODS: Formalin-fixed paraffin-embedded tissues from 153 patients who were pathologically diagnosed as having NSCLC were collected from November 2017 to October 2019. Both DNA/RNA-based NGS and RNA-based RT-PCR were used to detect EML4-ALK fusion. For samples with discordant ALK status results, fluorescence in situ hybridization (FISH) or Sanger sequencing was used to further confirm the ALK status. RESULTS: In total, 124 samples were successfully analyzed using both NGS and RT-PCR. For 118 samples, results were consistent between NGS and RT-PCR, with 25 reported as ALK fusion positive and 93 as ALK fusion negative, achieving a concordance rate of 95.16%. Among the six samples with disconcordant results, five were positive using RT-PCR but negative using NGS, and one was positive using NGS but negative using RT-PCR. Four of six cases with disconcordant results (three RT-PCR positive and one NGS positive) were successfully validated using either FISH or Sanger sequencing. CONCLUSIONS: Compared with NGS, RT-PCR appears to be a reliable method of detecting EML4-ALK fusion in patients with NSCLC.

Stress-induced premature senescence activated by the SENEX gene mediates apoptosis resistance of diffuse large B-cell lymphoma via promoting immunosuppressive cells and cytokines.

BACKGROUND: The underlying cause of relapsed and refractory (r/r) diffuse large B-cell lymphoma (DLBCL) is usually related to apoptosis resistance to antitumor drugs. The recent years have provided lots of evidence that tumor cells may undergo stress-induced premature senescence (SIPS) in response to chemotherapy, but how SIPS affects lymphoma cells remains inconclusive. METHODS: Fifty-two DLBCL patients, including 6 newly diagnosed (ND), 17 complete remissions (CR), and 29 (r/r), were enrolled in this study. We used a senescence-associated-ß-galactosidase (SA-ß-Gal) staining kit for senescence staining. Suppressive immune cells including regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) were detected by flow cytometry (FCM). Secreted cytokines were measured by ELISA Kit and SENEX gene expression was detected by a quantitative real-time polymerase chain reaction. We used 40 nM doxorubicin to induce the SIPS model of DLBCL in vitro. Apoptosis and proliferation activity of senescent LY8 cells were respectively detected by FCM and CCK8. SENEX gene was silenced by RNA interference. RESULTS: The proportion of senescent lymphoma cells was significantly increased in r/r DLBCL patients, concomitant with increased Treg, MDSC, and various secreted cytokines with proinflammatory and immunosuppressive effects. The SENEX gene was significantly elevated in the SIPS model. Senescent DLBCL cells had good antiapoptotic ability and proliferative activity accompanied by increased immunosuppressive cytokines. Interestingly, when we silenced the SENEX gene in the DLBCL cell line, the results were the opposite to the above. CONCLUSION: SIPS activated by the SENEX gene mediates apoptosis resistance of r/r DLBCL via promoting immunosuppressive cells and cytokines.

Chemoselective Hydrosilylation of the α,ß-Site Double Bond in α,ß- and α,ß,γ,δ-Unsaturated Ketones Catalyzed by Macrosteric Borane Promoted by Hexafluoro-2-propanol.

The B(C6F5)3-catalyzed chemoselective hydrosilylation of α,ß- and α,ß,γ,δ-unsaturated ketones into the corresponding non-symmetric ketones in mild reaction conditions is developed. Nearly 55 substrates including those bearing reducible functional groups such as alkynyl, alkenyl, cyano, and aromatic heterocycles are chemoselectively hydrosilylated in good to excellent yields. Isotope-labeling studies revealed that hexafluoro-2-propanol also served as a hydrogen source in the process.

The prognostic value of tumour-associated macrophages in Non-Hodgkin's lymphoma: A systematic review and meta-analysis.

Tumour-associated macrophages (TAMs) play an important role in the tumour environment and were reported to be associated with poor prognosis in several tumours. However, the prognostic significance of TAMs in Non-Hodgkin's Lymphoma (NHL) remains controversial. Consequently, we aimed to evaluate the relationship between subpopulations of TAMs and clinical outcomes in NHL patients. We did a comprehensive search of the PubMed, elsevier ScienceDirect, and Cochrane databases and extracted hazard ratio (HR) and their corresponding 95% confidence intervals (95% CIs) from eligible studies. Pooling total effect value by the stata statistical software and analysing correlation of TAMs with overall survival (OS) and progression-free survival (PFS). Furthermore, subgroup analysis and sensitivity analysis were also conducted. We deemed eleven studies, including 1211 NHL patients. Our study demonstrated that high-density CD68+ TAMs are associated with poor OS (HR: 1.17; 95% CI, 0.81-1.54; P = .000) and poor PFS (HR: 1.15; 95% CI, 0.63-1.67; P = .000) compared with low-density CD68+ TAMs in the tumour microenvironment. Similarly, high-density CD163+ TAMs can also predict poor OS (HR: 1.52; 95% CI, 1.11-1.92; P = .000) and shorter PFS (HR: 1.52; 95% CI, 0.73-2.30; P = .000). In addition, the high CD163+ /CD68+ TAMs ratio is significantly correlated with poor OS (HR: 3.59; 95% CI, 0.77-6.40; P = .013). However, in our subgroup analysis, high-density CD68+ TAMs in the tumour microenvironment is associated with better OS (HR: 0.75; 95% CI, 0.41-1.09; P = .000) in NHL patients treated with rituximab chemotherapy. Our results suggest that TAMs are a robust predictor of outcomes in NHL.

Stress-Induced Premature Senescence Promotes Proliferation by Activating the SENEX and p16INK4a/Retinoblastoma (Rb) Pathway in Diffuse Large B-Cell Lymphoma

Objective: Cellular senescence has been thought to be an important barrier to tumor formation. Recent studies have shown that stress-induced premature senescence (SIPS) can promote partial tumor invasion, but how SIPS affects diffuse large B-cell lymphoma (DLBCL) remains inconclusive. This study aimed to address that issue. Materials and Methods: The immunophenotype of the LY8 cell line was measured with flow cytometry. SIPS induced by tert-butyl hydroperoxide (tBHP) was detected by senescence ß-galactosidase staining. Cell proliferation was analyzed with CCK8 and expression levels of ARHGAP18 (SENEX gene-encoding protein), p16/p21, and Rb/pRb were measured with western blot. LY8 cells were transfected with SENEX-SiRNA/NC and verified by western blot. Results: Our results suggested that the immunophenotype of the LY8 cell line is CD19-, CD20-, and CD10-positive and the immunoglobulin light chain is the kappa type. The cellular senescence model of DLBCL could be successfully induced by 30 µM tBHP. ARHGAP18, p21, p16, and Rb protein levels were significantly increased but the level of pRb expression was decreased in the SIPS group compared with other groups. Meanwhile, the proliferation rate was increased in the SIPS group more than other tBHP groups. Furthermore, the expressions of p21 and p16 were significantly decreased in the SENEX-SiRNA group compared with the negative control group. Conclusion: SIPS formation activates ARHGAP18 and the p16/Rb pathway and promotes DLBCL cell proliferation. Furthermore, SENEX activates the p16 pathway in DLBCL. SIPS promotes proliferation by activating SENEX and the p16/Rb pathway in DLBCL. SENEX-related SIPS may serve as an important target for relapsed/refractory DLBCL therapy.

Compound Astragalus and Salvia miltiorrhiza extract inhibits hepatocarcinogenesis via modulating TGF-ß/TßR and Imp7/8.

Compound Astragalus and Salvia miltiorrhiza extract (CASE) is a Chinese herbal formula consisting of astragalosides, astragalus polysaccharide and salvianolic acids extracted from Astragalus membranaceus and Salvia miltiorhiza. Previous studies by our group have demonstrated that CASE effectively suppresses diethylinitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats via modulating transforming growth factor ß/Mothers against decapentaplegic (TGFß/Smad) signaling. To further elucidate the mechanism of CASE, the effects of CASE on TGF-ß1, the serine/threonine kinase receptors of TGF-ß [TGF-ß receptor type-I (TßRI) and TßRII] and karyopherins [Importin 7 (Imp7) and Imp8], which are crucial for TGF-ß/Smad signaling in fibro-hepatocarcinogenesis, were assessed in the present study using in vivo (DEN-induced HCC in rats) and in vitro [TGF-ß1-stimulated rat myofibroblasts (MFBs) and HepG2 cells] models of fibro-hepatocarcinogenesis. Hematoxylin and eosin staining revealed that CASE may suppress inflammatory reactions and fibrosis in HCC as well as increasing the differentiation of HCC cells. Positive TGF-ß1 staining was increased in HCC nodule areas and in adjacent normal liver tissues in DEN-treated rats, while TßRI staining was increased only in normal adjacent liver tissues. The elevated expression of TGF-ß1, TßRI and TßRII was suppressed by CASE. CASE treatment also reduced glutathione S-transferase P 1 and Imp7/8 protein expression in fibro-hepatocarcinogenesis. In vitro experiments confirmed that CASE was able to decrease the expression of TßRI and TßRII in TGF-ß1-stimulated MFBs and HepG2 cells. These results indicate that the anti-HCC effect of CASE may be achieved by mediating TGF-ß/TßR and Imp7/8 protein expression, suggesting that CASE has multiple targets in HCC treatment.

Cloning and characterization of GST fusion tag stabilized large subunit of Escherichia coli acetohydroxyacid synthase I.

There are three acetohydroxyacid synthase (AHAS, EC 4.1.3.18) isozymes (I, II, and III) in the enterobacteria Escherichia coli among which AHAS I is the most active. Its large subunit (LSU) possesses full catalytic machinery, but is unstable in the absence of the small subunit (SSU). To get applicable LSU of AHAS I, we prepared and characterized in this study the polypeptide as a His-tagged (His-LSU) and a glutathione S-transferase (GST)-tagged (GST-LSU) fusion protein, respectively. The results showed that the His-LSU is unstable, whereas the GST-LSU displays excellent stability. This phenomenon suggests that the GST polypeptide fusion tag could stabilize the target protein when compared with histidine tag. It is the first time that the stabilizing effect of the GST tag was observed. Further characterization of the GST-LSU protein indicated that it possesses the basic functions of AHAS I with a specific activity of 20.8 µmol min(-1) mg(-1) and a Km value for pyruvate of 0.95 mM. These observations imply that introduction of the GST fusion tag to LSU of AHAS I does not affect the function of the protein. The possible reasons that the GST fusion tag could make the LSU stable are initially discussed.

MAPK inhibitors modulate Smad2/3/4 complex cyto-nuclear translocation in myofibroblasts via Imp7/8 mediation.

Mitogen-activated protein kinase (MAPK) pathway-dependent linker phosphorylation of Smad2/3 and subsequent formation of Smad2/3/4 complex and its nuclear translocation are crucial for dysregulated transforming growth factor beta (TGF)-ß/Smad signaling in liver fibrosis. Abrogation of this critical step of TGF-ß/Smad signaling leading to liver fibrosis could provide new insights for future therapy, but the mechanisms remain incompletely understood. In pursuit, we investigated the subcellular expression and nuclear trafficking of the rate limiting Smad2/3/4 complex in exogenous TGF-ß1-stimulated myofibroblasts (MFBs) using three MAPK-specific inhibitors. Our results showed that exogenous TGF-ß1 stimulation of MFBs produced both increased protein expression and nuclear translocation of phosphorylated (p)-Smad2C/L, oncogenic pSmad3L, Smad4, importin7/8 (Imp7/8), and plasminogen activator inhibitor (PAI)-1 (Protein and mRNA), while decreased Smad7 protein expression. However, the MAPK-specific inhibitors differentially reversed these observations; for instance, ERK-specific inhibitor blocked the expression and nuclear translocation of pSmad2C/L, while both JNK and p38-specific inhibitors blocked the expression and nuclear translocation of pSmad2C/L and oncogenic pSmad3L. The MAPK-specific inhibitors had no significant effect on the total protein expression of Smad4, but rather significantly blocked its nuclear translocation. All the MAPK-specific inhibitors restored Smad7 expression and also decreased Imp7/8 and PAI-1 (Protein and mRNA) expression. Evidently, the MAPK-specific inhibitors blocked Smad2/3/4 complex formation via restoration of inhibitory Smad7 expression and blockade of Smad3L phosphorylation, while they blocked nuclear translocation of Smad2/3/4 complex through inhibition of Imp7/8 leading to decreased PAI-1 (Protein and mRNA) expression.

Compound Astragalus and Salvia miltiorrhiza extracts modulate MAPK-regulated TGF-ß/Smad signaling in hepatocellular carcinoma by multi-target mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus membranaceus Bunge (Leguminosae) and Salvia miltiorrhiza Bunge (Lamiaceae) are two important Chinese herbs with a long history of extensive ethnobotanical usage in the treatment of liver-related diseases over many centuries. Presently, these two herbs are being used either as a single herbal formulation or a composite formula for the treatment of liver related conditions. In response, recent studies on these two herbs have focused on elucidating their mechanisms of action, particularly with regards to their anti-hepatocarcinogenic effects. Previously, we have reported that Compound Astragalus and Salvia miltiorrhiza extract (CASE), a synergized composite extract from Astragalus membranaceus and Salvia miltiorrhiza ameliorates liver fibrosis and hepatocellular carcinoma (HCC) by modulating the TGF-ß/Smad pathway. Meanwhile, MAPK activation and MAPK-dependent linker phosphorylation of Smad2/3 and their preferential nuclear import are crucial for overall oncogenic role of TGF-ß/Smad signaling in HCC. To elucidate further, we studied the effect of CASE on the MAPK pathway and how it affects MAPK-dependent regulation of TGF-ß/Smad signaling using both cell and animal models of HCC. MATERIALS AND METHODS: We used immunofluorescence and western blot techniques to monitor effect of CASE on the activation of the MAPKs (pERK, pJNK and pp38) in TGF-ß1-stimulated hepatic stellate cells (HSCs), HepG2 cells and also diethylnitrosamine (DEN)-induced HCC in rats. Also phosphorylation and subcellular distribution of pSmad2/3, Smad4 and Imp7/8 in TGF-ß1-stimulated HSC and HepG2 cells were monitored. The expression of pERK, pJNK, pp38 and PAI-1 gene were monitored by using western blot technique. The effect of CASE on domain-specific phosphorylation of Smad2/3 and their subcellular distribution, and the expression of Smad4 and its subcellular distribution in TGF-ß1-stimulated HSCs and HepG2 cells were evaluated by using immunofluorescence technique. And the expression of Imp7/8 and their subcellular distribution were assessed by both immunofluorescence and western blot techniques, while PAI-1 gene expression was assessed by western blot RESULTS: In vitro, CASE in a concentration-dependent manner increased the expression of pp38 but decreased the expression of pERK and pJNK; however, in vivo, CASE in a dose dependent manner decreased the expression of pERK, pJNK as well as pp38. Also, CASE concentration dependently inhibited pSmad2C/L, pSmad3L, Smad4, Imp7/8 and their nuclear import; it had no effect on pSmad3C in HepG2 cells; significantly decreased PAI-1 gene expression in both in vitro and in vivo. CONCLUSIONS: CASE blocked MAPK activation, MAPK-dependent linker phosphorylation of Smad2/3, Smad4 expression, Imp7 expression and their nuclear import leading to significant down-regulation of PAI-1 gene expression; further highlighting the multi-target anti-HCC effect of CASE and its potential drug candidature.